Project Summary: The human body is a compilation of complex ecosystems that rely on bacteria. Phage infections that perturb these microbial communities have recently been implicated in a wide range of human disorders. Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of an RNA-guided gene regulation system that protects bacteria from phage infection and controls the expression of virulence factors. The long-term goal of our research is to understand the impact of CRISPR-mediated immune systems on the evolution and ecology of human-associated microbial communities. In Pseudomonas aeruginosa, CRISPR loci are transcribed and processed into small RNAs that are incorporated into the Csy complex, a large multi-subunit ribonucleoprotein required for protection against bacteriophages. However, there is a critical gap in our understanding of how the Csy complex finds invading nucleic acids and how these molecular beacons recruit effector nucleases (i.e., Cas3) to target nucleic acids for destruction. Recently, these CRISPR RNA-guided surveillance systems have been repurposed for programmable genome engineering in a wide variety of eukaryotic cells and model organisms that were previously recalcitrant to genetic manipulation. In this proposal we use a combination of biochemical (Aim 1) and structural (Aim 2) strategies to determine the mechanisms of CRISPR-Cas RNA-guided detection of foreign DNA by the adaptive immune system from P. aeruginosa (Type I-F) and determine how virally encode suppressors subvert this immune system. Results from these experiments will provide mechanistic insights that contribute to our long-term goal and have significance implications in the near-term application of these emerging tools for precise genome engineering.